1. Field of the Invention
Protein binding assays have become of increasing importance in the diagnosing of diseased states, the monitoring of the administration of drugs, and the determination of trace amounts of organic compounds unrelated to human health. Protein binding assays are particularly applicable in the determination of specific compounds which are present at concentrations of 10.sup.-6 M or less, particularly where they are present in a mixture of other compounds having similar properties.
Protein binding assays depend upon the ability of labeling an analyte, where the label provides a detectible signal, and where binding of the receptor to the labeled analyte permits discrimination between bound and unbound label. The presence of the receptor can either allow for a mechanical separation of bound and unbound labeled analyte or can affect the label in such a way as to modulate the detectible signal. The former situation is normally referred to as heterogeneous and the latter as homogeneous, in that the latter technique avoids a separation step.
In developing protein binding assays, there are a number of considerations. Considerations related to the choice of label include the sensitivity it provides, synthetic problems, stability, its sensitivity to changes in environment and the like. Other considerations are the purity required for the analyte and/or analyte receptor in preparing the reagents and in the assay. Additional considerations are the effect of varying analyte on the synthetic procedures, the properties of the label, and the sensitivity and accuracy of the assay. By having an assay technique which is generally applicable to a wide variety of analytes and is not significantly affected by variation in analyte, the preparation of reagents and the performance of the assay can be readily adapted to new and varying analytes.
2. Description of the Prior Art
Badley, "Fluorescent Probing of Dynamic and Molecular Organization of Biological Membranes," Modern Fluorescence Spectroscopy, Vol. 2 Ed. E. L. Wehry, Plenum Press, N.Y. 1976 and Kanaoka, Angew. Chem. Int. Ed. Engl. 16, 137 (1977) discuss fluorescent probes in biological systems. Wu et al, Biochemistry, 16, 3936 (1977), Harris, Chemistry and Physics of Lipids 19, 243 (1977) Smolarsky et al, J. of Imm. Meth., 15, 255 (1977) and Waggoner and Stryer, Proc. Nat. Acad. Sci. 67, 579 (1970) describe the use of fluorescent probes with biological membranes. Uemura, et al, Biochemistry 13, 1572 (1974), Alving and Richards, Immunochemistry 14, 373 (1977), Geiger and Smolarsky, J. Imm. Meth., 17, 7 (1977), Inoue and Nojima, Chem. Pharm. Bull. 16, 76 (1968) and Tamamura, et al, Japan J. Exp. Metd. 41, 31 (1971) describe antibody phospholipid interactions.
U.S. Pat. Nos. 3,850,578 and 3,887,698, and the references cited therein, teach the use of liposomes in assays where complement mediated lysis of the liposome as a result of antibody binding to an antigen bound to the liposome results in release of stable free radicals contained in the liposome.